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Raphaël
Restriction digest , 2h at 37°C
We want to amplify this DNA, so the idea is to cut the plasmid once to obtain a linear fragment that can be easily amplified with our primers.
- mRBS/TetA(C)+RBS/LuxI: 5µl miniprep + 1µl B4 + 0.1µl BSA + 3µl water + 1µl AlwNI
- wRBS/TetA(C)+RBS/LuxI: 5µl miniprep + 1µl B4 + 0.1µl BSA + 3µl water + 1µl AlwNI
- stRBS/TetA(C)+RBS/LuxI: 5µl miniprep + 1µl B4 + 0.1µl BSA + 3µl water + 1µl AlwNI
PCR
Primers : VF2 + VR
- mRBS/TetA(C)+RBS/LuxI
- wRBS/TetA(C)+RBS/LuxI
- sRBS/TetA(C)+RBS/LuxI
- mRBS/TetA(C)+RBS/LuxI cut by AlwNI
- wRBS/TetA(C)+RBS/LuxI cut by AlwNI
- sRBS/TetA(C)+RBS/LuxI cut by AlwNI
-> PCR tubes stored overnight at -20°C.
Ligation for 1h30 at 25°C
- wLac+LuxI : 1µl 1/2 dilution + 4µl + 1µL buffer + 4µL H20 + 0.5µl ligase
- sLac+LuxI : 1µl 1/4 dilution + 4µl + 1µL buffer + 4µL H20 + 0.5µl ligase
Control with only LuxI has already be done yesterday.
Transformation in Turbocells
With today's ligations:
Control :
Overnight cultures
From glycerols :
- wRBS/LacZα
- sRBS/LacZα
- RBS/LuxI
- pLux
- attC-t-attC-mRFP
- PmM-LuxR
- PmW-LuxR
- wTet-LuxI
- mTet-LuxI
- sTet-LuxI
From Aleksandra's transformations :
- 1x PmW-LuxR-attC
- 3x pLux-LuxI
- 4x PsS-attC + pBad-Int
- 4x PsM-attC + pBad-Int
- 4x PsW-attC + pBad-Int
- 4x PmS-attC + pBad-Int
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Memo cell
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